Analyte detection

ABSTRACT

The present invention relates to analyte detection test systems, including test systems for the oral detection of analytes in saliva. The present invention also provides compositions and methods for storing multiple assay tests and compositions and methods for measuring the concentration of analytes in a sample.

The present invention claims priority to U.S. Provisional ApplicationSer. No. 60/243,854 filed Oct. 27, 2000 and is a Continuation-in-PartU.S. patent application Ser. No. 09/393,552, filed Sep. 17, 1999 andU.S. patent application Ser. No. 09/698,306, filed Oct. 27, 2000, eachof which is hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to analyte detection test systems,including test systems for the oral detection of analytes in saliva. Thepresent invention also provides compositions and methods for storing anddistribution of multiple assay tests and compositions and methods formeasuring the concentration of analytes in a sample.

BACKGROUND OF THE INVENTION

Advances in detection technologies have made it possible to detect awide variety of substances in tissue and fluid samples from variousorganisms. For example, tests are available for qualitative andquantitative detection of glucose, proteins, illicit drugs, cancermarkers, cholesterol, pathogens, and other materials in human tissue andfluid samples.

However, many of the available tests are too expensive, cumbersome,complex, or dangerous for routine or frequent usage. For example, manyof the tests rely on electronic equipment that is too complex orexpensive for use by individuals outside of a laboratory or clinicalsetting. Additionally, many individuals have an aversion to certaintypes of sample testing procedures, such as blood and urine testing, andwould not be willing to self-administer such assay tests.

The art is in need of detection assay tests for the detection of a widevariety of analytes that adequately combine ease of use, small size,speed, accuracy, low cost, durability (e.g., temperature stability andshelf life), safety and interpretability, and that are designed forwidespread distribution and use. Furthermore, delivery systems forpackaging, storing, carrying, preserving, and otherwise maintainingtests are needed to provide adequate ease of use, discrete use,accessibility, and durability of tests.

SUMMARY OF THE INVENTION

The present invention relates to analyte detection test systems,including test systems for the oral detection of analytes in saliva. Thepresent invention also provides compositions and methods for storingmultiple assay tests and compositions and methods for measuring theconcentration of analytes in a sample.

For example, the present invention provides analyte detection assaytests for use in detecting the presence of an analyte in saliva. In someembodiments, the tests are configured for oral use (i.e., at least aportion of the assay test is placed into the mouth and contacted withsaliva). In some preferred embodiments, the tests are in the form of atest strip. In some embodiments, the test strip comprises an absorbentreaction pad at one end. In some preferred embodiments, the reaction padcontains reactants that create a detectable signal in the presence of ananalyte. In some such embodiments, the reaction pad is configured toproduce a detectable signal during or after the placement of thereaction pad into the mouth to collect saliva samples. In some preferredembodiments, the detectable signal comprises the formation of a color.In other preferred embodiments, the detectable signal comprises a changein color.

The present invention also provides a system comprising a plurality oftest assays for analyzing a sample for the presence of an analyte. Insome embodiments, the system comprises a plurality of assay tests withina delivery system, wherein the delivery system is configured to preventthe assay tests from being exposed to the environment. In someembodiments, the delivery system is configured to dispense the pluralityof assay tests individually (e.g., one at a time). In some embodiments,the delivery system is configured to dispense the plurality of assaytests individually without exposing the remaining assay tests (i.e., thetests that have not yet been dispensed) to the environment. In somepreferred embodiments, the delivery system comprises a desiccant. Insome embodiments, the delivery system comprises a desiccant entrainedpolymer storage container. In some embodiments, the delivery system isentirely lacking a desiccant.

The present invention further provides a system comprising a diagnosticdevice for analyzing saliva for the presence of an analyte, with thediagnostic device comprising: a solid support; one or more collectionsites attached to a first portion of the solid support, wherein thecollection site is configured to collect a saliva sample; one or morereaction sites (e.g., containing agents that produce a detectable signalin the presence of an analyte) attached to a second portion of the solidsupport, wherein the reaction sites produce a detectable signal in thepresence of the analyte; wherein the solid support, collection site, andreaction sites are contained within a single device (i.e., a “lateralflow” device). In some embodiments, no solid support is provided (i.e.,the test is made of only a collection site and reaction agents). In someembodiments, the collection site and reaction sites may be in contactwith one another (e.g., an absorbent material layered onto or integratedwith reaction agents). Thus, in some embodiments the first portion andsecond portion of the solid support may define partially or entirelyoverlapping regions of the solid support. In some embodiments of thepresent invention, multiple collection sites and multiple reactionssites are used. The plurality of collection sites find use, for example,in detecting different threshold concentrations of analyte (e.g., afirst collection site that detects 0.4% of analyte in saliva and asecond collection site that detects 0.8% of analyte in saliva),different detectable readouts (e.g., different colors or a firstcollection site that shows a color and a second collection site thatproduces a symbol, shape, or word), different read-out formats (e.g., afirst collection site that uses an on/off readout and a secondcollection site that uses a gradient readout), different detectionpurposes (e.g., detection versus indicator [to test if the assay isworking properly] or detection of different analytes) and the like.

In some embodiments of the present invention, the diagnostic devicecomprises a thickness X cm, a width Y cm, and a length Z cm, whereinX*Y*Z is less than 12 cm³, preferably less than 2 cm³ and morepreferably less than 1 cm³, although larger and smaller dimensions arealso contemplated by the present invention. In some embodiments, thethickness is 1.5 millimeters or less, the length is 5 centimeters orless, and the width is 1.25 centimeters or less. In other embodiments,the solid support comprises plastic. In yet other embodiments, thecollection site comprises an absorbent material.

The present invention provides a variety of analyte detection systems.For example, the present invention provides assay tests for use indetecting analytes including, but not limited to, alcohol, glucose,ketones, cancer markers (e.g., prostate-specific antigen [PSA],epidermal growth factor receptor [EGFR], cancer antigen CA 15-3),cortisol, serotonin, 5-hydoxytryptophane, methadone, cocaine,cannabinoids (e.g., 11-carboxy-Δ⁹-tetrahydocannabinolic acid), opiates,caffeine, phenytoin, primidone, carbamazepine, antibodies, pathogens(e.g., P. gingivalis, Chlamydia organisms, Streptococcus organisms,organisms that cause common infectious diseases such as the flu,measles, etc., Bacillus anthracis and other organisms that may be usedin biological warfare or terrorism, etc.), melatonin, insulin, DHEAsulfate, aldosterone, testosterone, progesterone, andostenedione,estriol, estrone, urea, uric acid, ammonia, calcium, cholesterol,lactoferrin, growth factors (e.g., EGF, NGF, IGF-1), haliperidol,theophylline, cotinine, estradiol, salicyclic acid, acetaminophen,nitrazepam, clobazam, amphetamine, quinine, lithium, antibiotics (e.g.,penicillin and tetracycline), vitamins, minerals, toxins, anti-oxidants,monosodium glutamate (MSG), components of food products (e.g., peanutsand/or tree nuts), proteins and nucleic acids (e.g., DNA and RNA),including host and non-host (e.g., pathogenic) proteins and nucleicacids.

In some embodiments, the reaction site comprises an enzyme the reactsdirectly or indirectly with the analyte to initiate a reaction resultingin the generation of a detectable signal. In some embodiments, thereaction site further comprises one or more competitors, wherein the oneor more competitors are configured to prevent the reaction site fromproducing the detectable signal until the one or more competitors aresubstantially depleted or otherwise prevent the detectable signal frombeing substantially detectable unless a threshold concentration ofanalyte is present in a sample. It is contemplated that, in someembodiments, multiple competitors are used, each with a differentthreshold level, such that the reaction site produces detectable signalsat two or more particular concentrations of test samples. However, itshould be noted that, in some embodiments, multiple threshold levels areobtained with the use of a single competitor or no competitors. Indeed,any desired detection configuration can be used. For example, a firstdetection event may occur at a desired threshold level of analyte,followed by a gradient detection read-out above the threshold level(e.g., a first detected color is observed above a concentration of0.04%, followed by a gradual increase in a color from concentrationsabove 0.04%). In some embodiments, the reaction site further comprisesone or more stabilizers (e.g., compounds that increase the shelf-life ofthe reaction site in response to moisture, light [e.g., ultra-violetlight], air, and the like). In yet other embodiments, the reaction sitecomprises two or more reaction components, wherein the two or morereaction components of the reaction site are separated by one or morebreakable barriers. In some embodiments, the reaction site is enclosedin a protective encasement.

In some embodiments, the enzyme used in the reaction site is an oxidaseor reductase that results in the production of oxidized and reducedreaction products. The oxidized or reduced reaction products result inthe generation of a detectable signal through a subsequentoxidation/reduction reaction (e.g., a reaction that results in a colorchanged cause by a change in the oxidation state of a chromogen). Thus,in some embodiments, any analyte containing a chemical moiety capable ofundergoing a change in oxidation state (e.g., analytes containingalcohol, ketone, aldehyde, and/or carboxylic acid groups) finds use withthe present invention. The present invention provides non-toxic,non-irritant, and/or non-carcinogenic colorimetric detection systems foruse in the reaction site, wherein the color is produced in response toan enzyme (e.g., oxidases, peroxidases, dehydrogenases) that generatesoxidized and reduced reaction products (e.g., hydrogen peroxide, NADP⁺,etc.). These detection systems find use as oral assay tests.

In some embodiments, the assay test employ one or more antibodies thatbind to an antigen, wherein the antigen is the analyte to be detected orwherein the antigen is detectable (e.g., generated) when the analyte tobe detected is present in a sample. In some preferred embodiments, acolorimetric system is used to indicate the binding between the antibodyand the antigen. These detection systems find use as oral assay tests.

In some embodiments of the present invention, the first and secondportions of the solid support are separated by a hinge. In otherembodiments, the first and second portions of the solid support areseparated by a breakable barrier. In yet other embodiments, thecollection site is slidingly attached to the solid support. In someembodiments, the collection site and reaction site are provided at thesame location.

In some preferred embodiments, the system further comprises a protectiveencasement, wherein the diagnostic device is enclosed in the protectiveencasement.

In some preferred embodiments of the present invention, the diagnosticdevice further comprises a second reaction site attached to a thirdportion of the solid support, wherein the second reaction site producesa second detectable signal, with the second detectable signal indicatinga sufficient volume of the test sample (e.g., saliva sample).

In some embodiments of the present invention, the detection test assaycomprises a “test strip” with a thickness X cm, a width Y cm, and alength Z cm, wherein X*Y*Z is less than 12 cm³, preferably less than 2cm³ and more preferably less than 1 cm³, although larger and smallerdimensions are also contemplated by the present invention. In someembodiments, the thickness is 0.5 millimeters or less, the length is 6.5centimeters or less, and the width is 5 millimeters or less.

The present invention further provides a system comprising a pluralityof test assays for analyzing a sample for the presence of an analyte,said system comprising a plurality of assay tests within a deliverysystem, said delivery system preventing the assay tests from beingexposed to the environment and wherein the delivery system is small(e.g., wallet sized, pocket sized, credit card sized). In someembodiments the small delivery system comprises a width (at the widestportion) of X cm, a length (at the longest portion) of Y cm, and athickness (at the thickest portion) of Z cm, wherein X*Y*Z is less than100 cm³ (e.g., 30 cm³ or less, 20 cm³ or less, 10 cm³ or less). In somepreferred embodiments, the delivery system is flat (e.g., comprising oneor more flat panels). In some such embodiments, the ratios of X:Z andY:Z are greater than 20:1 (e.g., greater than 30:1, greater than 40:1).In some embodiments, X is 6 cm or less, Y is 8.5 cm or less, and Z is 2mm or less. In preferred embodiments, the delivery system is configuredto dispense the plurality of assay tests individually.

In some embodiments of the present invention, the system furthercomprises a delivery system, whereby the delivery system comprises oneor more compartments capable of storing one or more of the diagnosticdevices. In preferred embodiments, the delivery system comprises two ormore compartments, each compartment accessible (e.g., independentlyaccessible) to allow use of one or more tests without exposing tests inother compartments. In some embodiments, the delivery system furthercomprises one or more protective encasements capable of enclosing thediagnostic devices in the one or more compartments. In yet otherembodiments the delivery system further comprising one or more placards.

The present invention also provides a delivery system comprising one ormore first packages comprising one or more compartments and a secondpackage containing the one or more first packages. For example, in oneembodiment, the delivery system comprises one or more assay tests, twoor more first packages comprising one or more compartments, wherein theone or more assay tests are contained in the first package; and a secondpackage, wherein the two or more first packages are contained in thesecond package. For example, in some embodiments, the second packagecomprises a thin folded delivery system. In some embodiments, the foldeddelivery system comprises a single fold, wherein the first package isaffixed to or contained in a portion of the delivery system such that,in folded form, the first package is enclosed within the folded deliverysystem. In yet other embodiments, the folded delivery system comprises apocket, wherein the first package is contained within the pocket. Inother embodiments, the folded delivery system comprises two or morefolds. For example, in some embodiments, the folded delivery systemcomprises two folds to provide a three-panel container.

In a particularly preferred embodiment, assay tests are contained infirst packages wherein the first packages comprise a first wall and asecond wall and wherein each wall comprises at least one layer. In someembodiments, multiple layers are provided. In some embodiments threelayers are provided where the inner-most layer comprises an interiorheat-sealed protective layer (e.g., a plastic layer), a intermediate“barrier” layer (e.g., a foil, polymer, or polymer film [SARAN, BARAX]layer), and an outer layer (e.g., a paper, cardboard, or polymer layer).In some embodiments, four layers are provided. For example, in someembodiments a “tie” layer is provided between the barrier layer andouter layer (e.g., a plastic or polymer [polyethylene] layer). The firstand second wall are connected at the edges to form an interior openingin which the assay test is enclosed (e.g., sealed). In some embodiments,the outer surface of each wall further comprises diagrams, text, orother written materials (e.g., instructions, warning, logo, etc.). Insome embodiments, the first packages are contained in second packages.In preferred embodiments, the second package is approximately the sizeand shape of a credit card. In one preferred embodiment, the secondpackage is made of a first wall and a second wall, wherein the secondwall is sealed to the first wall along three edges, forming an openingon one end of the second package. The first packages are insertable andremovable through the opening. In particularly preferred embodiments,the first wall of the second package is transparent to allow viewing ofthe contents (e.g., viewing of written materials on the first packagescontained within the second package). In other preferred embodiments,the second package is made of plastic. In yet other preferredembodiments, the second package contains two or more first packages(e.g., to allow users to access assay tests on more than one occasion).

In other embodiments, the second package comprises a first and secondwall connected by a hinge along one edge of the first and second walls.Assay tests are attached to the inner surface of the first wall (e.g.,enclosed in a pouch contained on the inner surface of the first wall).When the hinge is closed, the assay tests are enclosed between the firstand second walls. When the hinge is opened, the assay tests areaccessible.

In yet another preferred embodiment, the delivery system furthercomprises a flat solid support and one or more first packages (eachcontaining one or more assay tests in one or more compartments) attached(e.g., glued) to the flat solid support. In a preferred embodiment, thefirst packages are attached to the solid support in such a manner thatthe assay tests are accessible without removing the first package(s)from the solid support.

The present invention also provides a delivery system for storing assaytests, comprising one or more (e.g., two or more) compartmentsconfigured to contain assay tests. In some preferred embodiments, thedelivery system further comprises a plurality of protective encasementscapable of enclosing the assay tests in the compartments. In otherpreferred embodiments, the delivery system comprises a thickness, awidth, and a length, wherein the thickness is 2 millimeters or less, thelength is 6.0 centimeters or less, and/or the width is 8.5 cm or less.In yet other preferred embodiments, the delivery system has a thicknessless than 1 millimeter, a length less than 8.5 centimeters, and/or awidth less than 5.6 cm. In yet other preferred embodiments, the deliverysystem is the approximate size and shape of a standard credit card. Inyet other preferred embodiments, the delivery system further comprisesone or more placards.

While the present invention is not limited by the materials used in thedelivery system, in some preferred embodiments, the delivery system ismade of plastic or a paper or cardboard material. In some embodiments,the paper or cardboard material comprises laminated paper or cardboard.

The present invention further provides a delivery system for storingassay tests, comprising one or more (e.g., two or more) compartments andone or more assay tests, wherein the one or more assay tests arecontained within the one or more compartments. In preferred embodiments,the delivery system comprises two or more compartments, each containingone or more assay tests. In some preferred embodiments, the deliverysystem further comprises a plurality of protective encasements, whereinthe protective encasements enclose the one or more assay tests in thecompartments. In other preferred embodiments, the delivery systemcomprises a width (at the widest portion) of X cm, a length (at thelongest portion) of Y cm, and a thickness (at the thickest portion) of Zcm, wherein X*Y*Z is less than 100 cm³ (e.g., 30 cm³ or less, 20 cm³ orless, 10 cm³ or less). In some preferred embodiments, the deliverysystem is flat (e.g., comprising one or more flat panels). In some suchembodiments, the ratios of X:Z and Y:Z are greater than 20:1 (e.g.,greater than 30:1, greater than 40:1). In some embodiments the thicknessis 2 millimeters or less, the length is 5.5 centimeters or less, and/orthe width is 8.25 cm or less. In yet other preferred embodiments, thedelivery system has a thickness less than 1 millimeter, a length lessthan 8.5 centimeters, and/or a width less than 5.5 cm. In yet otherpreferred embodiments, the delivery system is the approximate size andshape of a standard credit card. In yet other preferred embodiments, thedelivery system further comprises one or more placards. In otherembodiments, the delivery system comprises plastic.

In some preferred embodiments, the delivery system comprises aprotective storage container comprising a round or oval vial with athickness, a width, and a length. While the present invention is notlimited by the dimensions of the container, in preferred embodiments,the thickness is X millimeters or less, the length is Y centimeters orless, and the diameter is Z cm or less wherein X*Y*Z is less than 15cm³, preferably less than 10 cm³. In some embodiments, the thickness is5 millimeters or less, the length is 5 centimeters or less, and thediameter is 2.5 cm or less. In other embodiments, the delivery system isa round or oval vial made of a thermo-plastic polymer with an entraineddesiccant that removes moisture from the interior of the container. Insome embodiments of the present invention, the system further comprisesa delivery system, whereby the delivery system comprises a protectivestorage container having one or more compartments capable of storing oneor more of the assay tests. In preferred embodiments, the storagecontainer has a cap providing an air tight seal and connected by a hingeof the same said polymer material wherein said cap, hinge and containerare molded as one piece. In still more preferred embodiments the storagecontainer is made of a hard thermo-plastic polymer with an entraineddesiccant allowing for easy accessibility without jeopardizing thereaction means to environmental moisture or humidity.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a top view of an assay test made with a hinge that allowsone end of a test to be folded onto the other.

FIG. 2 shows the three main components that make up the assay test inFIG. 1.

FIG. 3 shows a top view of a delivery system that stores multiple assaytests and is made with a hinge that allows the entire delivery system tobe opened.

FIG. 4 shows a top view of the delivery system in FIG. 3 when closed.

FIG. 5 shows an individual putting one end of an assay test from FIG. 1into their mouth.

FIG. 6 shows how a hinge allows one end of an assay test from FIG. 1 tobe folded onto another.

FIG. 7 shows a top view of an assay test from FIG. 1 where one end hasbeen folded onto the other.

FIG. 8 shows an assay test made with a hinge that allows two pieces fromone end of an assay test to be folded around the other end.

FIG. 9 shows an assay test made with a sliding mechanism that allows aportion from one end of an assay test to be slid around the other end.

FIG. 10 shows an assay test similar to the assay test in FIG. 1 butconstructed of two main components instead of three.

FIG. 11 shows a delivery system that stores multiple assay tests, and ismade with five hinges that allow five compartments in the deliverysystem to be opened.

FIG. 12 a shows an assay test from FIG. 1 and how it fits into aprotective encasement.

FIG. 12 b shows a delivery system that stores multiple assay tests andis made with multiple compartments that are each covered with aremovable protective encasement.

FIG. 13 a shows an assay test similar to the assay test in FIG. 8, butwhich comprises a secondary chamber containing additional reactioncomponents.

FIG. 13 b shows an assay test similar to the assay test in FIG. 9, butwhich comprises a secondary chamber containing additional reactioncomponents.

FIG. 13 c shows an assay test similar to the assay test in FIG. 1, butwhich comprises a secondary chamber containing additional reactioncomponents.

FIG. 13 d shows an assay test similar to the assay test in FIG. 13 c,but which comprises two main components instead of three.

FIG. 13 e shows the top view of an assay test similar to the assay testin FIG. 13 c except that two chambers containing additional reactioncomponents are located on the opposite end of the assay test from thechamber containing additional reaction components in FIG. 13 c.

FIG. 13 f shows a side view of the assay test in FIG. 13 e.

FIG. 14 shows one embodiment of the delivery systems of the presentinvention.

FIG. 15 shows one embodiment of the delivery systems of the presentinvention.

FIG. 16 shows one embodiment of the delivery systems of the presentinvention.

FIG. 17 shows one embodiment of the delivery systems of the presentinvention.

FIG. 18 shows an assay test strip in one embodiment of the presentinvention.

FIG. 19 shows a folded delivery system in one embodiment of the presentinvention.

GENERAL DESCRIPTION OF THE INVENTION

The present invention relates to assay test systems, includingcompositions and methods for storing multiple assay tests andcompositions and methods for measuring the presence of or concentrationof analytes in a sample. In preferred embodiments, the present inventionprovides:

-   -   1) An assay test that comprises a single device so that it is        easy to use. In preferred embodiments, the assay test is also        small, fast, accurate, inexpensive, safe, easy to read and        decipher, and durable; and    -   2) A delivery system that stores multiple assay tests so that        the assay tests can be accessed on one or more occasions. In        preferred embodiments, the delivery system makes assay tests        both easy to carry and durable. In some preferred embodiments,        the delivery system comprises a protective storage container        providing a cap with an air tight seal and a plug, sleeve,        pouch, liner, or other material comprising an entrained        desiccant that removes moisture away from the contents of the        container. In other preferred embodiments, the delivery system        comprises a folded structure, wherein assays test are attached        or enclosed in an interior portion of the folded structure. In        preferred embodiments, the delivery system is small and portable        to allow ease of handling.

Preferred embodiments of the present invention provide tests and systemsthat facilitate wide-spread use of assay tests by individuals. Forexample, the present invention provides assay tests that are containedwithin a single device and are easy to use, small, fast, accurate,reliable, inexpensive, easy to read and decipher and durable. Inpreferred embodiments, multiple tests are contained in a delivery systemsuch that one or more tests can be accessed and used at separate times.

Thus, in some embodiments, the assay test of the present inventioncomprises a single device so that it is easy to use. In preferredembodiments, the assay test is small so that it is easy to carry. Insome preferred embodiments, the assay test works fast so people do nothave to wait long before obtaining the results. In other preferredembodiments, the assay test is accurate so individuals can makedecisions based on correct information. In yet other preferredembodiments, the assay test is reliable so people know when it isfunctioning properly. In some embodiments, reliability is provided byassay tests that have undergone sufficient quality control andassessment to provide highly accurate and consistent results. In someembodiments, the assay test includes an indicator to identify, upon use,if the test is reliable. In some preferred embodiments, the assay testis inexpensive so that it can be afforded easily. In still otherpreferred embodiments, the assay test is durable so that it can behandled easily without breaking or becoming damaged. Unlike currentlyavailable detection devices, the assay tests of the present inventioncombine these desired features into a single, easy to use test thatsignificantly facilitates self-detection and assessment of analytes.

The present invention further provides delivery systems that a) storemultiple assay tests so that they can be accessed on one or moreoccasions (e.g., on one or more separate days, weeks, or months), b) insome embodiments, make assay tests durable and easy to access and carry,and c) in some other embodiments, provide placards for instructions,warnings, labels, and other text or diagrams. As mentioned above, thedelivery systems of the present invention store multiple assay tests sothat multiple assay tests can be accessed on a single occasion or on twoor more distinct occasions. This flexibility is important for severalreasons. For example, because individuals may use assay tests onseparate occasions, the delivery system stores a sufficient quantity oftests to last an individual a period of days, weeks or months, therebydiminishing the need to continually replenish assay test supply.Additionally, because individuals may use more than one tests on a givenoccasion, for example, to determine if their analyte concentration hasdropped over time, the delivery system stores multiple assay tests.

In preferred embodiments of the present invention, the delivery systemmakes assay tests easy to carry so that individuals can easily anddiscreetly put the assay tests in their pockets, wallets, or purses foruse in situations away from home. In other preferred embodiments, thedelivery system ensures the durability of the assay tests by enclosingthem in a protective hard container. In some preferred embodiments, thecontainer comprises a plastic-polymer container with an entraineddesiccant. Such a protective container assures that the assay tests donot break or spoil due to a breakdown or degradation of the biosensor,enzymes, antibodies, antigens, colorimetric agents, or other reactionagents. In yet other preferred embodiments, the delivery system makesassay tests easy to access so that removal of the assay test from thedelivery system can be conveniently accomplished, even by impairedindividuals. Thus, in some embodiments of the present invention, anassay test is dispensed upon opening the delivery system, while theremaining tests are maintained in the delivery system (e.g., maintainedso as not to be exposed to the environment). In some preferredembodiments, the delivery system provides large placards so thatinstructions, labels, warnings, or other text or diagrams are easy tonotice and read. The assay tests, first package, or second package mayalso contain such information.

The present invention further contemplates assay tests and deliverysystems that provide advantages for distribution of the tests andsystems to individual consumers by one or more secondary parties (i.e.,parties other than the consumer). For example, it is contemplated thatthe assay tests are provided to consumers by another party (e.g., arestaurant, bar, university, insurance company, employer, governmentagency, etc.). In such embodiments, it is contemplated that multipleassay tests are provided to the consumer so that testing can occur onmore than one occasion to avoid distribution each time the consumerneeds the test. Thus, in some embodiments of the present invention, itis desired to have delivery systems comprising multiple assay tests thatcan be accessed on one or more occasions. In other embodiments, it isdesired to have a protective container made of a thermoplastic polymerwith an entrained desiccant such that excess moisture is removed fromthe tests contained therein.

In some embodiments of the present invention, materials other than theassay tests are further included in or on the delivery systems. Forexample, in some embodiments, the delivery system or assay testcomprises an image or text associated with a company, agency, orindividual other than the provider of the assay test in order to obtainthe positive image associated with the assay tests of the presentinvention (i.e., co-branding). The co-branding may be provided on thesurface or interior of the delivery systems, may be included onmaterials attached to the delivery system, or may otherwise beassociated with the delivery system. In embodiments of the presentinvention where multiple assay tests are provided in a delivery systemfor use over a period of time, the co-branding provides constant andlong-term advertising for the entity providing the co-branding since theconsumer is in possession of the delivery system on their person forextended periods of time.

In other embodiments of the present invention, the delivery systemfurther comprises information or safety materials attached to, within,on, or otherwise associated with the delivery systems. Such materialsinclude, but are not limited to, information for transportation services(e.g., taxis, buses, etc.) and emergency services (e.g., police,hospital, etc.). In some embodiments, the materials comprise items thathelp individuals pass the time (e.g., while waiting for an analyte levelto increase or drop) including, but not limited to, puzzles, games,Internet access devices, etc (i.e., “time-consuming materials”—materialsother than the assay test or delivery system that can be used to occupyan individual's time for minutes to hours). In other embodiments, phonecards (e.g., pre-paid phone cards) or dial-in numbers are provided toallow the individual to arrange transportation or pass time. In stillfurther embodiments, the materials comprise rebates or coupons forproducts, and/or samples of a product. In yet other embodiments, thematerials comprises information related to awareness of physical,mental, and/or social problems related to the analyte.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below:

As used herein, the terms “assay test system,” “assay test,” and“diagnostic device” refer to any system capable of determining, eitherquantitatively or qualitatively, the presence of or concentration of ananalyte in a sample. Such assay test systems include both detectionassay tests themselves (e.g., devices or combinations of devices thatcontain sample collection and analyte detection capabilities) and anyassociated “delivery systems” (i.e., systems used to store, transport,and maintain assay tests and other items). In some preferredembodiments, the “test assay” comprises a simple test strip containing areactive site at one end, such that the reactive site provides adetection element in the presence of analyte when exposed to a samplesuspected of containing analyte.

As used herein, the term “sample” is used in its broadest sense. In onesense it can refer to a saliva sample. In another sense, it is meant toinclude a specimen or culture obtained from any source, includingbiological and environmental samples. Biological samples may be obtainedfrom animals (including humans) and encompass fluids, solids, tissues,and gases. Biological samples include blood products (e.g., plasma andserum), saliva, urine, lachrymal fluid, cell lysates and the like.Environmental samples include environmental material such as surfacematter, soil, water, and industrial samples. These examples are not tobe construed as limiting the sample types applicable to the presentinvention.

As used herein, the terms “reaction means,” “reaction agent,” and“reaction site” refer to compositions that provide for a reaction. Forexample, reaction means include, but are not limited to: enzymes,cofactors, and buffers for enzymatic reactions; ligands, analytes, orbiosensors; and any other composition that facilitates a reaction. Forexample, where the analyte is an alcohol (e.g., methanol, ethanol etc.),in one embodiment of the present invention, the reaction means comprisesan alcohol dehydrogenase, NAD(P)H and/or NADH cofactors, a diaphorase,and a chromogen for colorimetrically detecting the presence of alcoholin a sample. In another embodiment, the reaction means comprises analcohol oxidase. The term “biosensors” refers to any sensor that ispartially or entirely composed of biological molecules. In a traditionalsense, the term refers to “an analytical tool or system consisting of animmobilized biological material (such as enzyme, antibody, whole cell,organelle, or combination thereof) in intimate contact with a suitabletransducer device which will convert the biochemical signal into aquantifiable electrical signal” (Gronow, Trends Biochem. Sci. 9: 336[1984]). However, as used herein, the term biosensor is not limited tothe incorporation or association with transducer devices. The presentinvention contemplates biosensors with and without transducer devices.

As used herein, the term “immobilization” refers to the attachment orentrapment, either chemically or otherwise, of material to anotherentity (e.g., a solid support, substrate, or surface) in a manner thatrestricts the movement of the material.

As used herein, the terms “solid support,” “solid substrate,” and “solidsurface” refer to any material that provides a solid or semi-solidstructure with which another material can be attached. Such materialsinclude smooth supports (e.g., metal, glass, plastic, silicon, andceramic surfaces) as well as textured and porous materials. Suchmaterials also include, but are not limited to, gels, rubbers, polymers,and other non-rigid materials. Solid supports need not be flat. Supportsinclude any type of shape including spherical shapes (e.g., beads).Materials attached to solid support may be attached to any portion ofthe solid support (e.g., may be attached to an interior portion of aporous solid support material). For example, solid supports include, butare not limited to, plastic, ceramic, paper, cardboard, or metalsupports structures for supporting or enclosing collection sites,reactions means, or other compositions. In some embodiments, solidsupports may further comprise other materials (e.g., desiccants).

As used herein, the term “collection site” refers to a portion of acomposition capable of collecting a sample. Collection sites include,but are not limited to, hydrophilic pads, porous membranes, films,patches, polymers (e.g., silicone, rubber, acrylics), and absorbentmaterials. For examples, collections sites include, but are not limitedto, polypropylene, polyethylene, polystyrene, polyester, polyacrylatesand methacrylates, polyacrylamide, polyisobutylene (synthetic rubbers),starch, and cellulose (See e.g., U.S. Pat. No. 5,585,273, hereinincorporated by reference in its entirety). The term “absorbentmaterial” includes, but is not limited to, cotton or other thinfiber-based material, paper (e.g., filter paper), cloth, sponge, andother absorbent materials.

As used herein, the term “alcohol metabolizing enzymes” refers to anyenzyme capable of reacting with an alcohol substrate. Alcoholmetabolizing enzymes include but are not limited to alcoholdehydrogenases and alcohol oxidases.

As used herein, the term “enzyme” refers to molecules or moleculeaggregates that are responsible for catalyzing chemical and biologicalreactions. Such molecules are typically proteins but can also be shortpeptides, RNAs, or other molecules.

As used herein, the term “competitor” refers to an any means capable ofreducing the rate of a reaction. In some embodiments, competitorsinclude, but are not limited to competing substrates that compete withanother substrate for access to an enzyme active site. The competingsubstrate may have greater or lessor affinity for the active site thanthe other substrate. In other embodiments, competitors include, but arenot limited to trapping agents that prevent a substrate from reactingwith an enzyme or prevent a reaction product from being detected.

As used herein, the term “substantially depleted” refers to a competingsubstrate that has reacted with an enzyme to such a degree that othersubstrates are capable of accessing the enzyme at significant levels(e.g., detectable levels).

As used herein, the term “sol-gel” refers to preparations composed ofporous metal oxide glass structures. Such structures can have biological(e.g., enzymes) or other material entrapped within the porousstructures. The phrase “sol-gel matrices” refers to the structurescomprising the porous metal oxide glass with or without entrappedmaterial. The term “sol-gel material” refers to any material prepared bythe sol-gel process including the glass material itself and anyentrapped material within the porous structure of the glass. As usedherein, the term “sol-gel method” refers to any method that results inthe production of porous metal oxide glass. In some embodiments,“sol-gel method” refers to such methods conducted under mild temperatureconditions. The terms “sol-gel glass” and “metal oxide glass” refer toglass material prepared by the sol-gel method and include inorganicmaterial or mixed organic/inorganic material. The materials used toproduce the glass can include, but are not limited to, aluminates,aluminosilicates, titanates, ormosils (organically modified silanes),and other metal oxides.

As used herein, the term “direct colorimetric detection” refers to thedetection of color changes without the aid of an intervening processingstep (e.g., without conversion of a color change into an electronicsignal that is processed by an interpreting device). It is intended thatthe term encompass visual observing (e.g., observing with the humaneye).

As used herein, the term “chromophore” refers to molecules or moleculargroups responsible for the color of a compound, material, or sample.

As used herein, the term “aqueous” refers to a liquid mixture containingwater, among other components.

As used herein, the term “breakable barrier” refers to a barrier betweenchambers or wells that can be broken, for example, by bending,compressing, heating, snapping, twisting, or other disruptions, suchthat the contents of the chambers or wells have access to one another.

As used herein, the term “indicator” refers to a detectable signal thatindicates the introduction of sufficient sample to a reaction means fora desired (e.g., detectable and reliable) reaction to take place.

As used herein, the term “protective encasement” refers to a thincovering, wrapping or shielding comprising a material that acts toprotect a composition such as a reaction means (e.g., to extend theshelf-life of the reaction means).

As used herein, the term “communication facilitating agent” refers to anagent that allows an individual to initiate a communication with anotherindividual or with another entity. Communication facilitating agentsinclude, but are not limited to, phone cards, pre-paid dial-in phonenumbers, Internet access information, etc. that allow one to initiate acommunication with another party (e.g., a party capable of providing theindividual transportation).

As used herein, the term “low-moisture environment” refers toenvironments with a lower moisture content then the ambient environment.

As used herein, the term “sleeve,” when referring to delivery systems,refers to a pouch-like enclosure comprising at least two, preferablythree, sealed sides to provide an opening for the insertion, removal,enclosure, and storage of another item. In some preferred embodiments,the sleeve is contained on a credit-card sized delivery system (e.g.,approximately 9 cm×6 cm×2 mm). One embodiment of such a sleevecontaining delivery system is shown in FIG. 14. The sleeve portion ofthe delivery system may be made of any material, including, but notlimited to, plastic, paper, cardboard, and the like.

As used herein, the term “entrained” refers to materials that areintegrated, attached, or in fixed contact with other materials. Forexample, entrained desiccants are desiccants that are mixed withinanother material or attached or fixed to another material (e.g.,attached or fixed as a lining within a container).

As used herein, the term “time-consuming materials” refers to materialsother than assay tests or delivery systems that can be used to occupy anindividual's time (e.g., for minutes to hours). Such materials maycomprise objects or may be written material or other text. Such writtenmaterial may, is some embodiments, be included on assay tests ordelivery systems. Examples of time-consuming materials include, but arenot limited to puzzles, games, Internet access devices. Time consumingmaterials find use, for example, in passing time while waiting for one'sblood alcohol concentration to drop.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides systems for determining the presence ofor level of an analyte in a sample. For example, the present inventionprovides qualitative and quantitative detection assays that determine ifan analyte is present in a sample at or above one or more specificconcentration levels.

In preferred embodiments of the present invention, the test assaycomprises an oral test assay. In particularly preferred embodiments, thetest assay is an easy-to-use test strip comprising a reaction site atone end. In some preferred embodiments, the reaction site is place intothe mouth of an individual and is saturated with saliva. If the targetedanalyte is present in the saliva, a detectable signal is produced,indicating the presence of, or concentration of, the analyte. In somepreferred embodiments, the detectable signal is detectable by the humaneye (e.g., a viewable color change). However, the present inventionincludes detectable signals that are read by a device. Because thereaction site is placed in the mouth, it is preferred that components ofthe reaction site are non-toxic, non-irritant, non-carcinogenic, andotherwise do not produce any undesired or inconvenient physicalreactions in the user.

The following description highlights several preferred embodiments ofthe present invention. The present invention is not limited to theseillustrative examples. While the following description highlights theuse of test strips for oral use, it should be understood that the otherconfigurations are within the scope of the present invention. Thedetailed description is provided in the following sections: I) AssayTests; II) Delivery Systems; and III) Analytes.

I. Assay Tests

A. Description

The assay test is preferably small in size so that it can be easilycarried. For example, in some embodiments, the assay test forms a‘strip’ and is rectangular, flat, and thin, such that multiple assaytest strips can be stored in a delivery system that is convenient tocarry. An example of a test strip in one embodiment of the presentinvention is shown in FIG. 18.

In preferred embodiments, the components of the assay test of thepresent invention are contained within a single device so that it iseasy to use. The assay tests comprises three main components: a solidsupport, a collection site and a reaction means. In some embodiments,the assay tests comprise a desiccant material.

1. Solid Support

The assay test comprises a solid support providing both a handle or freeend to hold the test with as well as providing a substrate for othercompositions (e.g., collection site). In embodiments where the assaytest comprises a test strip, the solid support is typically a thinpaper, filter, or plastic material wherein one end of the strip providesthe free end to hold, while the other end of the strip contains thecollection site and reaction means. In some embodiments, the solidsupport comprises a desiccant material.

2. Collection Site

The assay test comprises a collection site for collecting a sample. Insome embodiments of the assay test, the collection site comprises anabsorbent material that can absorb a sample (e.g., a fluid such assaliva) from an individual. In some embodiments, the sample flows fromthe absorbent material to a reaction means (e.g., by diffusion), whilein other embodiments the collection site is physically introduced near areaction means such that the sample is introduced to the reaction means.In yet other embodiment, the reaction means and collection site are incontact with one another or are integrated. Several assay test formatsthat allow the introduction of the sample from an absorbent material toa reaction means are described below. In other embodiments of thepresent invention, a sample is directly introduced into a reaction meanswithout an absorbent material (e.g., by introduction of fluid into acollection site comprising a well or chamber).

In some embodiments, the assay test is provided with a stimulator,configured to stimulate saliva production in the mouth. Theseembodiments are employed, where it is desired or necessary to have largevolumes of saliva or where newly produced saliva provides a moreaccurate correlation to blood concentrations of analyte (e.g., forglucose detection). Any physical (e.g., a physical protrusion the entersthe mouth and provokes salivation), psychological (e.g., images orscents the stimulate salivation), or chemical (e.g., saliva stimulationtablets that do not interfere with the detection chemistry; flavors)methods for stimulating saliva production are contemplated.

3. Reaction Means

The assay test further comprises a reaction means for detecting thepresence of analyte in a sample. A wide variety of reaction means arecompatible with the present invention. Acceptable reaction means arethose that can be incorporated into the assay tests of the presentinvention and that can maintain a detectable signal in the presence ofanalyte. Reaction components that find use in the detection ofparticular analytes are described in Section III, below. In someembodiments, the reaction means comprises an enzyme that reacts with theanalyte, directly or indirectly, to generate a reaction product that isdirectly or indirectly detected. For example, enzymes that react withanalytes and produces an oxidized or reduced reaction product find usein the present invention. In some embodiments, the oxidized or reducedproduct is used is a reaction with a chromogen to produce a visiblydetectable color. In some embodiments, a series of reactions are used toamplify the signal. For example, reaction products of a first reactionare each used to generate multiple reaction products in a secondreaction. The reaction products of the second reaction are then used insubsequent reactions, eventually leading to the generation of detectableresponse.

In some embodiments, the reaction means provides a qualitativemeasurement of analyte concentration. In some embodiments, the presenceof a detectable signal indicates the presence of an analyte, withoutindicating a specific amount of the analyte. In other embodiments, thedetectable signal appears when a particular threshold analyteconcentration is present. While this indicates that the concentration ofanalyte is above a certain concentration, it does not provide aquantitative measure of actual analyte concentration. In someembodiments, the detectable signal (e.g., color) increases withincreasing analyte concentration. An estimate of analyte concentrationis made by comparing the level of detectable signal to a chart or tablerepresenting ranges of analyte concentration that correspond to anapproximate level of detectable signal. In some embodiments, thedetectable signal is measured to determine a quantitative amount analytein the sample. In some embodiments, one or more control test assays areused to assist in quantitative determination (e.g., a second test assayis used on a sample with known analyte concentration).

In other embodiments, of the present invention, a biosensor is used. Awide variety of biosensors find use in the reaction means of the presentinvention, including, but not limited to the biosensors described inU.S. Pat. Nos. 5,571,395, 5,792,621, 5,500,351, and 6,183,772,incorporated herein by reference in their entireties. In someembodiments, the biosensor employs an antibody. In some embodiments ofthe present invention, the reaction means provide a colorimetricresponse that intensifies with increasing concentrations of analyte(e.g., a gradient reading). In alternate embodiments, the reactionoccurs at one particular or multiple threshold levels, as desired (Seee.g., U.S. Pat. No. 5,032,506, incorporated herein by reference in itsentirety).

In some embodiments that require a series of chemical reactions to takeplace in sequence, the assay test further comprises multiple chambersfor separating, isolating, combining, or storing the reactioncomponents. For example, when a chemical is stored dry, but active onlyin aqueous solution, separate chambers store the chemical and aqueoussolution. Directly prior to or during use of the assay test, thecontents of the chambers are combined (e.g., by breaking a barrierseparating the separated components).

In some embodiments of the present invention, the reaction means isimmobilized to increase durability, accuracy, and ease of use. Forexample, in some embodiments the reaction means is immobilized on filterpaper, or another material, which allows transfer of the sample to thereaction means and provides a reflective surface for enhancedcolorimetric detection. The reaction means may also be immobilized inchambers or in gels. In some embodiments of the present invention, thereaction means is immobilized in a porous metal oxide matrix using thesol-gel method (See generally, Brinker and Scherer, Sol-Gel Science,Academic Press, San Diego [1995]). Sol-gel entrapment providescost-efficient, stable, accurate, reliable, consistent, and robustmaterials that can be produced in a variety of shapes and sizes. Theunique properties of sol-gel materials such as optical transparency,durability, and tailorable properties (e.g., porosity, surfacefunctionalization, thin films, and bulk materials) provide an idealmaterial for immobilization of colorimetric materials. The sol-gelprocess has been used for entrapping organic molecules such as dyes andproteins in silica gels (See e.g., Avnir, Accounts Chem. Res. 28: 328[1995]; Yamanaka et al., Am. Chem. Soc. 117: 9095 [1995]; Miller et al.,Non-Cryst. Solids 202: 279 [1996]; and Dave et al., Anal. Chem. 66:1120A [1994]).

In particularly preferred embodiments, the assay test further comprisesan indicator that comprises a second reaction means. The indicatorprovides a detectable signal indicating the introduction of sufficientsample to the first reaction means for a reaction to take place,ensuring the reliability of the assay test. Several of such preferredembodiments are described in detail below. For example, in oneembodiment of the present invention, the indicator is located at the endof a sample path, downstream of the first reaction means. The samplemust pass through the first reaction means before reaching theindicator. By providing the indicator with a second reaction means, apositive result with the indicator demonstrates that a sufficient amountof sample has been exposed to the first reaction means. In someembodiments, the indicator employs the same chemistry as the primaryassay reaction site, while in other embodiments scavengers are excludedand/or a different chromagen is used (independent of toxicity,irritability, or carcinogenicity if the indicator is not exposed to theuser).

In other preferred embodiments, the assay test comprises a protectiveencasement. In some embodiments, the protective encasement comprises amaterial such as foil and covers the reaction means. In suchembodiments, the protective encasement is automatically broken andreveals the reaction means when the user operates the assay test. Instill other embodiments, the protective encasement comprises a materialsuch as foil and surrounds the entire assay test. In such embodiments,the user opens the protective encasement to reveal the assay test beforeoperating the assay test.

B. Operation

In one preferred embodiment of the present invention, a protectivestorage container is opened to reveal an assay test. The assay testoperates by first saturating an absorbent material on one end of theassay test with a sample. Depending on the reaction means used in theassay test, the user waits for a period of time and interprets thedetectable signal produced by the reaction means. In embodiments thatemploy an indicator, to check the reliability of the assay test, theuser observes if enough sample was initially put on the absorbentmaterial by viewing a detectable signal from the indicator. The absenceof a detectable signal from the indicator demonstrates that not enoughsample was initially put on the absorbent material, and that the testmay not be reliable. Finally, the user checks their analyteconcentration by viewing a color change or other detectable signal(e.g., the appearance of a symbol such as a shape or word) from thereaction means. In some embodiments, to make the assay test easy todecipher, the user compares the color changes to pictorial and writteninstructions printed on the assay test or a delivery system.

In another preferred embodiment of the present invention, a protectiveencasement is opened to reveal an assay test. The assay test operates byfirst saturating an absorbent material on one end of the assay test witha sample. The user then, in one-step, either (1) folds or slides thissaturated end into a well on the second region of the assay test, or (2)folds or slides pieces from a second region of the assay test around oronto the saturated end. In preferred embodiments, the folding or slidingmotion is designed for quick and easy use. Detection is carried out asdescribed above.

In some preferred embodiments where an enzyme is used as part of thereaction means, the portion of the test assay containing the reactionmeans (e.g., a pad on a test strip) is maintained in the mouth of asubject for an extended period of time (e.g., for 5 seconds to severalminutes), as the enzymatic reaction proceeds at a higher rate at theelevated temperature in the mouth. This method is in contrast to methodswhere the test assay is briefly saturated with the saliva and thenincubated at room temperature until color development. Where theenzymatic reaction is allowed to occur in the mouth, the entire reactiontime from initial testing to detecting the presence or absence of acolor change is carried out in less time. The increased speed forreading the assay increases the likelihood that the assays will be used.

One embodiment of the alcohol concentration assay test of the presentinvention is illustrated in FIG. 1. The assay test is approximately 1.5mm in thickness, and has overall dimensions of roughly 5 cm×1.25 cm,although both larger and smaller dimensions are contemplated and can bedesigned, as desired (e.g., a thickness X cm, a width Y cm, and a lengthZ cm, wherein X*Y*Z is less than 12 cm³, preferably less than 2 cm³ andmore preferably less than 1 cm³).

FIG. 2 illustrates the assay test components of the assay test shown inFIG. 1. The assay test comprises three main parts, a base 16, a middle20, and a top 21. The base 16, middle 20, and top 21 are constructed ofa strong, durable material such as plastic, although a variety ofmaterials are contemplated by the present invention. In this figure, theattachments to the base 16 include a hinge 15, two filter avenues 18, awell covering 40, an absorbent material 42, and a reaction meansimpregnated on a thin sheet 14 for detecting the presence of alcohol ina sample. The hinge 15 is constructed in conjunction with the base 16 toform a single molded part. The hinge is made of a thin, flexiblematerial such as plastic, although a variety of materials arecontemplated by the present invention. The hinge 15 allows the wellcovering 40 to easily fold, snap, and lock onto the well 38 on themiddle section 20. The hinge 15 also allows the absorbent material 42 toeasily fold into the well 38. The filter avenues 18 allow the fluidsample from the absorbent material to travel up both sides of the base16. A material or body that draws a fluid sample such as filter paper orsmall capillary tubes is used to construct the filter avenues 18. Thewell covering 40 is constructed in conjunction with the base 16 to formone molded part. The absorbent material 42 is constructed of a materialthat absorbs and collects a desired fluid sample (e.g., saliva) such asa synthetic sponge or cotton fibers, although a variety of materials arecontemplated by the present invention. The sheet 14 comprises either:(a) a pre-established, fast, inexpensive, and accurate chemical reactionmeans which produces a controlled color change, (b) a pre-established,fast, inexpensive, and accurate biosensor which produces a controlledcolor change, or (c) any other accurate, inexpensive, and fasttechnology that reacts in the presence of analyte to produce acontrolled detectable signal (e.g., a color change).

The attachments to the middle 20 include a well 38 and a porous membrane34. The well 38 is constructed in conjunction with the middle 20 to formone molded part. The well 38 is made so that the absorbent material 42compresses to fit snugly inside. In addition, the well 38 is constructedso that the well covering 40 snaps and locks on top of the well 38. Themembrane 34 is located at the bottom of the well 38. It is made of aporous material that allows the fluid sample to pass but does not allowother debris to pass.

The attachments to the top 21 include a small window 22, large window30, and air hole 26. The two windows 22 and 30 are open or transparentspaces that allow the sheet 14 to be viewed through the middle 20. Theair hole 26 is a hole in both the top 21 and the middle 20 which allowsair to escape from the base 16.

II. Delivery Systems

A. Description

The present invention provides delivery systems for assay tests thatstore one or more assay tests so that assay tests can be accessed on asingle occasion or on two or more distinct occasions. In someembodiments, the delivery system also comprises a protective storagecontainer making assay tests both durable and easy to access, carry, anddistribute, and, in other embodiments, comprises placards that allowinstructions, labels, warnings or other text or diagrams to be easilynoticed and read. In still other embodiments, the delivery systemscomprises additional materials, including but not limited to,co-branding materials, phone cards, etc, as described above.

The delivery system of the present invention is preferably small in sizeso that it can be easily carried. For example, in some embodiments, thestorage container is rectangular, flat, and thin (e.g., shaped like acredit card), while in other embodiments the container is round, oval,or other shapes as shown in FIG. 17 (e.g., with a height ofapproximately 5 cm or less and a diameter of approximately 2.5 cm orless), such that individuals can easily and discreetly carry thedelivery systems in their pockets, wallets, or purses for use insituations away from home. In some embodiments, the delivery system isflat and comprises a folded structure. In one embodiment, the foldeddelivery system comprises a single fold (e.g., to panels connected by ahinged portion), such that, when folded, the assay tests are providedwithin the folded structure. The assay tests can be associated with thefolded in structure in any manner. For example, in some embodiments, theassay tests (directly or contained within a protective encasement) areaffixed to the inside of the folded structure by an adhesive. In otherembodiments, the assay tests are enclosed in a pocket. In some preferredembodiments, the delivery system comprises two folds and three panels.An example of such a structure is shown in FIG. 19. In some preferredembodiments, the delivery system, when folded, has a length of 8.5 cm orless, a width of 5.5 cm or less, and a thickness of 1 mm or less. Inpreferred embodiments, the assay tests are provided on a portion of thefolded structure such that opening of first flap exposes the assay test.The use of a three-panel delivery system provides six panel sides (i.e.,each of the three panels has a front and back side). Delivery systemswith multiple panels provide a surface area for the addition of text,figures, or attachment sites for additional materials. Such text,figures, and additional materials include, but are not limited to,branding, co-branding, instructions, information, or other attachedmaterials (e.g., phone cards, etc. as described above).

In some preferred embodiments, the delivery system is manufactured toincorporate a desiccant so that environmental moisture does not affectthe biosensors or otherwise impair the reaction means of the assay. Insome embodiments, a desiccant material is placed in a chamber of thedelivery system or attached to an interior surface of the container(e.g., lined in a plastic bottle or lined in a foil container). However,in preferred embodiments of the present invention, the desiccantmaterial is incorporated into the material of the delivery system (e.g.,the entire material, the walls, the bottom, the cap, etc.). In someembodiments, the inside of the container has a plug, liner, or sleevethat is made of an entrained desiccant that channels moisture away fromthe inside of the container and into the desiccant thereby keepingmoisture away from the tests. In preferred embodiments the desiccant isco-molded at the same time as the container allowing the desiccant toform a sleeve within the container and channeling moisture away from thecontents of the container. Methods for generating suchdesiccant-entrained polymers are described in U.S. Pat. Nos. 5,911,937and 6,080,350, as well as PCT publications WO 98/39231, WO 99/63288, WO99/62697, and WO 00/17259, each of which is incorporated herein byreference in their entireties. The presence of the desiccant in thematerial provides consistent drying capabilities throughout thecontainer so that each of a plurality of tests contained in thecontainer remain equivalently reliable over time. In some embodiments,the desiccant is not attached or associated with the delivery system(e.g., provided in an unattached pouch).

The present invention is not limited by the nature of the desiccant, orby the use of a desiccant. In some embodiments, the desiccant comprisesone or more chemical compounds that form crystals that contain water(e.g., anhydrous salts), compounds that undergo a chemical reaction withwater or moisture, and materials capable of moisture absorption throughphysical absorption (e.g., silica gels, molecular sieves, and naturallyoccurring clay compounds such as montmorillimite clay).

A number of container materials may be used in the generation ofentrained desiccants. For example, matrix based polymers of the presentinvention can be basically any functionalized thermoplastic includinganhydride or amine or acid or cyanate or isocyanate or hydroxyfunctionalized polymer. Examples of suitable matrix based polymers, asdescribed in U.S. Pat. No. 6,080,350, include polypropylene maleicanhydride, polyethylene maleic anhydride, polystyrene maleic anhydride,polyethylene acrylic acid, polyethylene-urethere, polyethylene-EVOH andpolyethylene-nylon. Other suitable thermoplastic materials includegrafted polyolefins, polycarbonates, polyamides, ethylene-vinyl acetatepartially hydrolyzed polymers, ethylene-methacrylate partiallyhydrolyzed polymer, grafted polyvinyl chloride, grafted polystyrene,polyester, polyester amide, polyacrylic partially hydrolyzed ester,acrylic, polyurethane and polyacetal or mixtures thereof. In someembodiments of the present invention, the desiccant containing materialcomprises channels to allow moisture to be eliminated by the entrainedmaterial. The channeling agent used in the present invention, asdescribed in U.S. Pat. No. 6,080,350, can be generally any hydrophilicmaterial. In one embodiment, the hydrophilic material is a polarcompound having at least two hydroxy groups. Suitable channeling agentsof the present invention include polyglycols such as polyethylene glycoland polypropylene glycol and mixtures thereof. Other suitable materialsinclude EVOH, glycerin, pentaerithritol, PVOH, polyvinylpyrollidine,vinylpyrollidone or N-methyl pyrollidone, with polysaccharide basedcompounds such as glucose, fructose, and their alcohols, and mannitolbeing suitable for the purposes of the present invention since they arehydrophilic compounds having numerous hydroxy groups.

The desiccant-containing delivery systems of the present invention areused to increase the shelf-life of the assay tests. Thus, when used inconjunction with the multiple-test-containing delivery systems of thepresent invention, the test can be accessed at different time periodsand still maintain functionality (i.e., the ability to accurately detectthe presence of alcohol in a sample). In some embodiments, tests may bestored and accessed for one month to two years or more and stillmaintain functionality (e.g., one month, two months, . . . one year, . .. two years, . . . ).

In some embodiments, no desiccant material is used at all. The presentinvention provides delivery systems and assay tests that allow the assaytests to remain functional over extended periods of time withoutdesiccation. Test systems without desiccant are manufactured lessexpensively. Thus, the desiccant-free test systems of the presentinvention provide an advantage over available desiccant-containingsystems.

In preferred embodiments, the delivery system acts as a storagecontainer and stores multiple assay tests so that one or more assaytests can be accessed on a single occasion or on two or more distinctoccasions. In some embodiments, the delivery system comprises a flatcredit-card shaped structure (e.g., a folded structure as describedabove). In some embodiments of the present invention, the deliverysystem comprises a thin box, an oval, round, or other shaped cylinder,or other desired shapes, that includes one or more compartments formultiple assay test storage. In preferred embodiments, the storagecontainer comprises a material such as hard plastic that protects theassay tests and increases their durability, while in other embodiments,the delivery system comprises a paper or cardboard-like material (e.g.,laminated paper). In particularly preferred embodiments, the deliverysystem is constructed so that it can be easily opened to access assaytests. In other embodiments the delivery system is made of a hardplastic polymer with an entrained desiccant so that moisture ischanneled away from the contents contained therein, maintaining a lowhumidity and preserving the viability of the reaction means of the assaytests. In other embodiments, removable protective encasements cover oneor more compartments.

Storing multiple assay tests so that they can be accessed on a singleoccasion or two or more distinct occasions has several benefits.Multiple assay tests allow individuals to use more than one assay teston a given occasion, for example, to determine if their analyteconcentration has increased or dropped over time. Additionally, becauseindividuals may use assay tests on separate occasions, the deliverysystem stores a sufficient quantity of tests to last an individual aperiod of days, weeks, or months; thereby diminishing the need tocontinually replenish assay test supply. For example, where the testsare provided to consumers by a party other than the consumer, thedistribution of the system by the secondary party is more efficient(e.g., requires less resources) if multiple tests are distributed at onetime rather than providing tests on separate occasions. In addition, insome embodiments, it is desirable for the delivery system to be durableso that the assay tests are not damaged during distribution from asecondary party to a consumer (e.g., distribution by mail).

In some embodiments of the present invention, the delivery system isdesigned to allow easy access to the assay tests. For example, tests maybe accessed by simply snapping open an air tight cap that covers one ormore chambers containing the tests. In preferred embodiments thedelivery system comprises a hard plastic polymer storage container withan entrained desiccant, thereby, protecting the assay tests inside fromenvironmental moisture. In other embodiments, tests may be accessed bysimply lifting a flap that covers one or more chambers containing thetests. Alternately, the tests may be directly accessed through anopening at one portion of a chamber. In yet other embodiments, thedelivery system comprises a folded structure (e.g., two flaps connectedby a hinge or three flaps connected by two hinges) whereby unfolding ofthe structure reveals one or more of the assay tests. In some of theseembodiments, exposure of the assay tests to the environment is increased(a negative consequence) in trade for easier access (i.e., the assaytests are not completely sealed from exposure to moisture, air, light[e.g., ultra-violet light], heat fluctuations, and the like). In someembodiments, the assay tests comprise one or more stabilizers thatincrease shelf-life in response to environmental exposure.

In some embodiments of the present invention, assay tests are containedin a first package. The first package may contain one or more tests. Inembodiments where the first package contains more than one test, thetest may be contained in one or more compartments in the first package.In some embodiments, the first package is sealed to protect the assaytests from the environment. In some embodiments, one or more of thefirst packages are contained in a second package. In preferredembodiments, two or more first packages are contained in a secondpackage which can be independently opened to gain access to the assaytest. The present invention contemplates delivery systems comprisingsuch first and second packages. In further embodiments, the deliverysystem provides placards so that instructions, labels, warnings, orother text or diagrams are easy to notice and read. In some embodiments,such materials are provided on the first or second packages or on theassay test itself.

In yet other embodiments, the delivery system stores multiple tests andis configured to dispense a single assay test without exposing theremaining tests to the environment. For example, in one embodiment, teststrips are provided in a ribbon form within the delivery systems,wherein the tests are connected to one another end to end with aserration creating the division point between test strips. In someembodiments, the ribbon is provided in a roll within the deliverysystem. In such embodiments, a small portion of the end test strip canbe exposed to the environment such that the reactive portion of saidtest strip is maintained within the delivery system without beingexposed to light or moisture from the outside environment. A user pullsthe end of the strip forward out of the device such that the reactiveportion of the desired test strip and the end of the next test stripemerges from the delivery system. The serration between the desired teststrip is then severed to release the desired test strip for use, leavingthe end (the non-reactive end) of the next test strip exposed for futureuse. In still other embodiments, the multiple test strips are notconnected to one another, but are still dispensed from the deliverysystem a single test strip at a time. In such embodiments, the useractivates a selector contained on the delivery systems which forces asingle test strip out of the delivery system while maintaining theremaining test strips within the protective environment of the deliverysystem (See e.g., U.S. Pat. No. 4,911,344, herein incorporated byreference in its entirety). In some embodiments, the test strip isdispensed by a pivoting cover having an internal finger portion thatpushes the topmost test strip of a stack of test strips outwardly fromthe top of the dispenser as the cover pivots (See e.g., U.S. Pat. Nos.4,171,753, 3,942,683, 3,845,882, 3,844,445, 3,565,284, 3,410,455, and2,853,206).

B. Operation

In one preferred embodiment of the present invention, the deliverysystem is operated by opening the delivery system. In some embodiments,a test is then removed for use. In other embodiments, a removableprotective encasement that covers a compartment of the delivery systemis peeled or folded back or otherwise opened or removed to reveal anassay test. The assay test is then removed for use.

One embodiment of the delivery system of the present invention isillustrated in FIGS. 3 and 4. The delivery system is rectangular, flat,and thin, similar in size and shape to a credit card, so that it is easyto carry in a wallet, pocket, or purse. The delivery system isapproximately 2 mm in thickness, and has overall dimensions of roughly5.5 cm×8.25 cm, although smaller or larger delivery systems can begenerated as desired. The components of the delivery systems in FIGS. 3and 4 are a compartment 44, hinge 45, locking mechanism 48, indentations56, and three placards 46, 50, and 54. The compartment 44 holds multipleassay tests and can hold fewer or more assay tests than are shown inFIG. 3. Assay tests are individually placed in a foil or otherprotective encasement 72, shown in FIG. 12 a, so they can be used onseparate occasions. In addition, multiple assay tests are individuallyplaced in protective encasements 72 so that the supply of assay testscan last an individual a period of weeks or months. Further, multipleassay tests are contained in the delivery system so that individualshave enough assay tests to determine if their analyte concentration hasdropped over time on one distinct occasion. Alternately, the compartment44 can be covered with a removable protective encasement, which can bepeeled back or otherwise removed to reveal an assay test as shown inFIG. 12 b.

As shown in FIGS. 3 and 4, the hinge 45 is constructed in conjunctionwith the delivery system to form one molded part. The hinge 45 is madeof a material such as plastic, although a variety of materials arecontemplated by the present invention, that allows the delivery systemto be easily opened and closed to access assay tests. The lockingmechanism 48 is constructed so that the delivery system closes tightlyto protect assay tests. In addition, the delivery system is constructedof a material such as hard plastic, although a variety of materials arecontemplated by the present invention, that will protect the assay testsand add to their durability. The indentations 56 are molded to protrudeinto the compartment 44 to limit the ability of the assay tests to moveand consequently become damaged while inside the delivery system.Finally, three placards 46, 50, and 54 allow instructions, labels, andwarnings to be easily noticed and read. Placard 50 refers to the frontof the top of the delivery system. Placard 46 refers to the back of thetop of the delivery system. Placard 54 refers to the back of the bottomof the delivery system.

Additional embodiments of the assay tests are shown in FIGS. 8, 9, 10,and 13 a-f. There are various possibilities to how the absorbentmaterial 42 is fitted into the well 38 in one easy step. In FIG. 8, thehinge 15 allows both the well 38 and well covering 40 to easily fold,snap, and lock around the absorbent material 42. In FIG. 9, a slidingmechanism 17 replaces the hinge 15 so that the well 38 and well covering40 easily slide on top of and tightly lock around the absorbent material42. There are also various possibilities to the number of partsnecessary to build an assay test. In FIG. 10, the top 21 is not used.Instead the middle 20 is colored such that the sheet 14 cannot be viewedexcept through the windows 30 and 22. In addition, there are variouspossibilities for the location of the reaction means. In FIGS. 13 a-f,the reaction means is located on a sheet 14 and/or in one or morechambers 31. In FIGS. 13 a-f, when an individual folds or slides theassay test to operate it, a protrusion 33 breaks open a chamber 31 andintroduces the chamber's contents to the sheet 14, thereby releasing ormixing the components of the reaction means.

An additional embodiment of the delivery system is shown in FIG. 11.There are various possibilities to constructing a rectangular, flat, andthin delivery system that provides multiple placards and stores testswhile making them easy to carry, easy to access on one or moreoccasions, and durable. In FIG. 11, there are five hinges 45. Each hinge45 breaks off to reveal separate compartments containing assay tests.

From the description above, a number of advantages of the assay testsystems of the present invention become evident. Because the assay testis contained within a single device, it is easy to use. For example, insome embodiments, the hinge on the assay test allows the test to beeasily used in one step. In other embodiments, the assay test comprisesa reaction means which relies on a chemical (e.g., enzymatic),biosensor, or other technology that provides the assay test with fastand accurate detection capabilities, lowers costs, and produces acontrolled color or other detectable change. In some embodiments, theassay test has an indicator that ensures reliability by allowing theuser to check if enough sample was put on the absorbent material. Also,in other embodiments, the large, easy to see window allows results to beeasily read, and the pictorial and written instructions that appear onthe assay test and/or delivery system allow results to be easilydeciphered and interpreted.

Because the assay test and the delivery system are small and haverelatively few parts, in some embodiments, the assay test and deliverysystem are inexpensive to manufacture. Because the delivery systemcomprises a rectangular, flat, and thin design, similar in size andshape to a credit card, in some embodiments, it is easy to carry in awallet, pocket, or purse. Because the delivery system stores multipleassay tests that are protected by their own protective encasements,assay tests are easy to access individually on one or multipleoccasions. In some embodiments, the hard material of which the deliverysystem is constructed protects the assay tests and adds to theirdurability. In yet other embodiments, the delivery system provides largeplacards that allow instructions, labels, and warnings to be easilynoticed and read.

In some embodiments of the present invention, the delivery systemoperates by first unlocking the locking mechanism 48. Next, a user foldsopen the delivery system and removes one assay test enclosed in aprotective encasement 72. The encasement 72 is then easily ripped openand an assay test is removed. As shown in FIG. 5, a user then saturatesan absorbent material 42 on one end of the assay test with a salivasample. Next, as shown in FIG. 6, the user, in one step, folds thissaturated end into a well 38 on the opposite part of the test. Thefolding motion is quick and easy. Depending on the technologyimpregnated on the sheet 14, the user waits a short period of time. Tocheck if enough saliva was initially put on the absorbent material 42,at the end of the waiting period, the user observes a color change orother detectable signal in the small circular window 22. The absence ofa change in the window 22 indicates that enough saliva may not have beeninitially put on the absorbent material 42, and the assay test shouldnot be used. Finally, the user checks if their saliva analyteconcentration level is at or above a specific level by viewing a colorchange or other detectable signal in the large, easy to read, octagonshaped window 30. To make the assay test easy to decipher, the usercompares the color changes or other detectable signals in the windows 22and 30 to pictorial and written instructions printed on the test 58, 62,66, and 68, and on the delivery system placards 46, 50, and 54.

In another embodiment of the present invention the delivery systemcomprises first and second packages. As shown in FIG. 14, a firstpackage 102 contains an alcohol concentration test 105. In someembodiments, the first package comprises multiple compartments, each ofwhich contain one or more alcohol concentration tests. The first package102 comprises a first wall 103 and a second wall 104. The walls may be asingle material or may comprises layers of different materials. In someembodiments, the walls comprise an inner layer (e.g., heat sealedplastic or polymer), an intermediate layer (e.g., a foil, polymer, orpolymer film [SARAN, BARAX] layer), and an outer layer (e.g., a paper,cardboard, or polymer layer), while in other embodiments, four layer areprovided including a tie layer (e.g., a plastic or polymer[polyethylene] layer) between the intermediate layer and outer layer. Insome embodiments, the first package is sealed, preventing exposure ofthe assay test to the environment. A second package 99 comprises a firstwall 100 and a second wall 101. The first and second walls are sealedalong three sides. The open end provides an opening for the insertion orremoval of one or more of the first packages 102 between the first wall100 and second wall 101. In preferred embodiments, the second package isthe approximate size and shape of a standard credit card. In somepreferred embodiments, the first or second wall of the second packagefurther comprises a thumb notch at the unsealed side to facilitate entryor removal of the first packages.

In another embodiment shown in FIG. 15, a first package 205 (asdescribed above for first package 102) is enclosed in a second package200. The second package 200 comprises a first wall 201 with an innersurface 203 and a second wall 202 with an inner surface 204. The firstwall 201 and second wall 202 are connected along one edge by a hinge206. The first package 205 is attached to the inner surface 204 of thesecond wall 202. When the hinge 206 is in the closed position, the firstpackage 205 is enclosed within the second package 200. When the hinge206 is in an open position, the first package 205 is accessible.

In another embodiment shown in FIG. 16 the delivery system 300 comprisesa solid support 301 and an assay test 305 enclosed within a package 302.In some embodiments, the package 302 comprises multiple compartments,each of which contain one or more assay tests. The package comprises afirst wall 303 and a second wall 304. The walls may be a single materialor may comprises layers of different materials. In some embodiments, thewalls comprise a heat sealed plastic inner layer, a foil intermediatelayer, and a paper outer layer. In some embodiments, the first packageis sealed, preventing exposure of the alcohol concentration test 305 tothe environment. The second wall 304 of the package 302 is attached(e.g., glued) to the solid support 301. In preferred embodiments, thedelivery system 300 is approximately the size and shape of a standardcredit card.

In still other embodiments of the delivery system of the presentinvention, the delivery system is a rectangular, oval or roundcylinder-like storage container, or other desired shape. In someembodiments, it is made of a hard plastic polymer, so that it is easy tocarry in a pocket or purse. In particularly preferred embodiments, thestorage container is approximately 2-3 mm in thickness, has a height of5 cm or less and a diameter (e.g., outer diameter) of approximately 2.5cm, although smaller or larger delivery systems can be generated asdesired. An example of one such configuration is shown in FIG. 17. Inthis figures, the container comprises a cap that forms an airtight sealwhen snapped onto the container. In some embodiments, the cap isattached to the container by a hinge. In still further embodiments, thecap is sealed by a locking mechanism so that the delivery system closestightly to protect assay tests and to provide an air tight seal toreduce the exposure of the tests to the outside environment. Suchdelivery systems find particular use with test assays that are in a teststrip format. For example, as shown in FIG. 18, in some embodiments, theassay test strip is approximately 0.5 mm in thickness, and has overalldimensions of roughly 6.5 cm×5 mm, although both larger and smallerdimensions are contemplated and can be designed, as desired.

In yet other embodiments, the delivery system is provided as a foldedstructure, as exemplified in FIG. 19. In some preferred embodiments, thedelivery system, when folded, has a length of 8.5 cm or less, a width of5.5 cm or less, and a thickness of 1 mm or less. In this figure thefolded structure has two folds and three panels, each panel having afront and back side. In some embodiments, the assay test is associatedwith a panel, such that it is exposed when the first flap is opened(e.g., the front of panel 1 or the back of panel 3 in FIG. 19). Theremaining panels find use for displaying or providing attachment orinsertion points for text, figures, or other desired materials (e.g.,co-branding and phone cards), as described above. In preferredembodiments, the delivery system is FIG. 19 is composed of paper orcardboard stock (e.g., laminated paper or cardboard stock). In yet otherpreferred embodiments, the delivery system, when folded, isapproximately the dimension of a credit card so as to facilitate its usein wallets or purses. While the folded delivery systems may not provideas much protection from the environment as sealed containers, in someembodiments, the folded delivery systems are inexpensive to produce,providing the appropriate combination of durability (e.g., using bothfolded delivery systems and attached foil packages that encase the assaytests to provide sufficient durability) and ease of use, costefficiency, and the ability to provide substantial co-branding,instructions, and other desired materials (e.g., pre-paid phone cards,material for passing time, etc.).

Accordingly, it is clear that the assay test system of the presentinvention comprises assay tests and delivery systems that have manysignificant advantages. In some embodiments, the assay test is containedwithin a single device so that it is easy to use. In some embodiments,the assay test is also small, fast, accurate, inexpensive, and durable.In addition, in some embodiments, assay test results are easy to readand easy to decipher using either the delivery system or the assay testitself. The assay test relies on either a chemical, biosensor, or otherdetection technology as a reaction means. The delivery system storesmultiple assay tests so that the assay tests can be easily accessed onone or more occasions. In some embodiments, the delivery system makesassay tests both easy to carry and durable. In other embodiments, thedelivery system provides placards for instructions, warnings, andlabels.

III. Analytes

The present invention provides detection assay tests for a wide varietyof analytes. In preferred embodiments, the present invention providesoral assay tests for measuring analytes in saliva samples. The assaytests find use in the detection of analytes including, but not limitedto, alcohol (e.g., for use by individuals in making decisions aboutwhether or not to operate a motor vehicle), glucose (e.g., qualitativeor quantitative tests for use by diabetics), ketones, cancer markers(e.g., prostate-specific antigen [PSA], epidermal growth factor receptor[EGFR], cancer antigen CA 15-3), illicit compounds (e.g., cocaine,cannabinoids [e.g., 11-carboxy-Δ⁹-tetrahydocannabinolic acid], opiates),caffeine, hormones (e.g., natural and synthetic hormones includingaldosterone, testosterone, progesterone, andostenedione, estriol,estrone, steroids, fertility markers, pregnancy markers), antibodies,pathogens (e.g., P. gingivalis, Chlamydia organisms, Streptococcusorganisms, etc.), growth factors (e.g., EGF, NGF, IGF-1), and othercompound including, but not limited to, cortisol, serotonin,5-hydoxytryptophane, methadone, phenytoin, primidone, carbamazepine,melatonin, insulin, DHEA sulfate, urea, uric acid, ammonia, cholesterol,lactoferrin, haliperidol, theophylline, cotinine, estradiol, salicyclicacid, acetaminophen, nitrazepam, clobazam, amphetamine, quinine,lithium, antibiotics (e.g., penicillin and tetracycline), vitamins,minerals (e.g., calcium), toxins, anti-oxidants, monosodium glutamate(MSG), components of food products (e.g., peanuts and/or tree nuts),proteins and nucleic acids (e.g., DNA and RNA), including host andnon-host (e.g., pathogenic) proteins and nucleic acids.

Configurations of detection assays are illustrated below for severaldifferent classes of analytes. It will be appreciated that theseconfigurations are applicable and adaptable to assay tests directed tothe detection of other analytes.

A) Alcohol

Many of the costs, risks, and penalties associated with alcohol-relatedaccidents could be prevented if individual alcohol consumers werecapable of making a self-assessment of their capacity to engage inpotentially dangerous activities. Although the inebriated individualoften bears the greatest risk of harm from alcohol-related incidents,they may not have the motivation, knowledge, materials, or ability totake effective steps in preventing undesired alcohol-related incidents.The present invention provides systems that addresses the unmet need ofproviding individuals with easy to use and readily accessible alcoholconcentration tests. The systems and methods of the present invention donot require the individual to incur substantial costs or expendsubstantial resources in obtaining and using the alcohol concentrationtests—greatly increasing the likelihood that the tests are used and theassociated benefits incurred.

In preferred embodiments, the assay tests are portable and durable andcan be carried and stored in a wallet or purse. The consumer may use thetest at home as a method of learning how to gauge physical symptomsassociated with particular blood alcohol concentrations. The alcoholconsumer may also read instructional and educational materials thataccompany the test, gaining a better understanding of the relationshipbetween alcohol impairment and accidents. In preferred embodiments, thetest is used following alcohol consumption as a method for aiding thedetermination of fitness to drive. If the test indicates blood alcoholconcentration (BAC) levels associated with risk of driving, the consumerchooses not to drive, reducing the risk of accidents. In someembodiments, a group of alcohol consumers are each tested to determinethe most suitable driver or drivers within the group. In otherembodiments, an alcohol consumer, upon receiving a result indicatingimpairment, waits for a time period and conducts subsequent testinguntil the results suggest fitness to operate a motor vehicle. In someembodiments, the assay tests are provided with time-consuming materialsto help occupy the consumers' time between testing events. In otherembodiments, information (e.g., taxi information) is provided to assistthe consumer in selecting a safe course of action if the test indicatesa lack of fitness.

In some embodiments of the present invention, alcohol providers such asbars, restaurants, and alcohol manufacturers and distributors providealcohol concentration tests to consumers. For example, in someembodiments, a restaurant may implement a wait-and-retest program (e.g.,providing multiple tests and educational information and/or incentivessuch as free non-alcoholic beverages until a suitable test result isobtained).

In preferred embodiments, the alcohol concentration assays tests arestable in the for at least one month, preferably for at least sixmonths, more preferably for at least one year, and most preferably forat least two years. For example, in some embodiments, the assay test istemperature stable and possesses a long shelf life (e.g., maintainsfunction for over a year at room temperature and for over three monthsat 104° F.).

A wide variety of reaction means are compatible with the presentinvention. In preferred embodiments, acceptable reaction means are thosethat can be incorporated into the assay tests of the present inventionand that can generate and maintain a detectable signal in the presenceof alcohol (e.g., methanol, ethanol, etc.). In some embodiments, thereaction means is selected and tailored to achieve desired reactionspeed, accuracy, reliability, cost, and durability. For example, avariety of chemical reactions that provide colorimetric detection ofethanol in a sample are described in U.S. Pat. Nos. 5,032,506,4,629,697, 4,642,286, 5,290,683, 5,589,349, 5,429,932, 5,429,931,5,416,004, 4,786,596, 4,810,633, 4,734,360, 5,525,481, 5,141,854,5,403,749, incorporated herein by reference in their entireties.

In preferred embodiments, the chemicals used at the reaction site arenon-toxic, non-irritant and/or are not carcinogenic. This presents aproblem because many chromogens cause toxic reactions, irritation or arecarcinogenic if used orally. In contrast, the present invention provideschemistries that are non-toxic, non-irritant and/or non-carcinogenic.Indeed, any non-toxic, non-irritant and/or non-carcinogenic chromogenfinds use with the present invention. A chromogen with unknown toxicitycan be tested for toxicity by exposing several doses of the material toa test subject (e.g., an animal or human) and detecting undesired toxicresponses. If no undesired toxic responses are observed when thechromogen is used at a functional (e.g., colorimetric) concentration,when exposed to the subject in a manner consistent with the methods ofthe present invention (e.g., placed in the mouth of a subject on acolorimetric test strip), then the candidate compound may be designatednon-toxic and incorporated into the test assays of the presentinvention. The protocol in Example 1 may be followed to determinewhether or not a candidate material for use in the reaction site of thetest assay is toxic/non-toxic or an irritant/non-irritant. To avoidcarcinogenicity, components of the reaction means are selected such thatthey are known not to have carcinogenicity (See e.g., CRC Handbook ofIdentified Carcinogens and Noncarcinogens). For compounds where thecarcinogenicity is unknown, testing can be conducted to determinewhether the candidate compound is, or is not, a carcinogen using anytechnique known in the art.

In some embodiments, the present invention provides potassium iodide(KI) as a chromogen. Assay tests containing potassium iodide were testedas non-toxic and non-irritant using the protocol in Example 1. Potassiumiodide is an approved food additive whose use at 0.01% in iodized saltis well recognized. The amount of potassium iodide used in an assay test(e.g., 133 micrograms) is equivalent to the amount contained in 1-2grams of iodized salt, and presents no safety concerns. Potassium iodidewas found to provide a stable, detectable, colorimetric chromogen foruse in the assay tests of the present invention. Potassium iodide alsoprovides a chromogen suitable for use in on/off type assay readouts.Embodiments employing non-toxic chromogens are described in detailbelow.

In preferred embodiments, the reaction site of the present inventionprovides an on/off readout if the alcohol concentration level of thesample is above a certain threshold concentration. For example, an assaytest designed to detect a sample concentration equivalent to a bloodalcohol concentration of 0.04% would not change color at concentrationsignificantly under 0.04% (off), but would change color atconcentrations at or above 0.04% (on). The on/off format can beaccomplished, for example, by the addition of competitive inhibitors orscavengers that prevent the colorimetric reaction unless the thresholdconcentration is reached, whereupon the competitor or scavenger isswamped out and the substrate is available to initiate the colorimetricreaction. Embodiments employing on/off reactions are described in detailin the example section below. In yet other embodiments, the detectionreadout is a gradient readout, wherein the color change graduallyincreases in intensity with an increase in alcohol concentration. Insome embodiments, both on/off and gradient detections readouts arecombined in a single assay. In some embodiments of the presentinvention, multiple collection sites and reaction sites are used. Theplurality of collections sites find use, for example, in detectingdifferent threshold concentrations of alcohol (e.g., a first collectionsite that detects 0.4% and a second collection site that detects 0.8%),different detectable readouts (e.g., different colors or a firstcollection site that shows a color and a second collection site thatproduces a symbol, shape, or word), different read-out formats (e.g., afirst collection site that uses an on/off readout and a secondcollection site that uses a gradient readout), different detectionpurposes (e.g., detection versus indicator or detection of differentanalytes) and the like.

In some preferred embodiments, stabilizers are used to make the assaytest durable. For example, in some embodiments of the present invention,the assay tests of the present invention remain functional for over ayear when maintained at room temperature and for over three months at104° F. This durability is enhanced when the assay tests are stored inthe delivery systems of the present invention. For example, in someembodiments, assay tests enclosed in a delivery system remain functionalfor over two years.

Certain illustrative chemistries for use at the reaction site areprovides below. The exemplary chemistries are not intended to limit thescope of the invention. In one embodiment of the present invention, thechemicals used at the reaction site are prepared in several submixesprior to combination and application on the desired solid support (e.g.,test strip). The first submix comprises water (RO/DI water) (0.48 kg),dextran (40.0 g), starch (7.50 g), and gelatin (6.25 g). The water isadded to a suitably sized vessel, followed by addition, with stirring,of dextran, starch, and gelatin. The components are stirred untilhomogenous (of even appearance). The solution will be milky inappearance. After the solution is homogenous, the mixture is heated toboiling (100° C.) for three minutes after the mix turns clear. Themixture is then cooled with stirring (ice should not be used). Themixture may become slightly cloudy after cooling.

The second submix comprises water (RO/DI water) (240 g), potassium EDTA(120 mg), citric acid (11.53 g), and potassium iodide (12.5 g),L-Cysteine, and sodium nitrite (the amounts of L-Cysteine and sodiumnitrite are dependent on the activity of the alcohol oxidase).L-Cysteine and sodium nitrite may be used at 290 mg/L and 130 mg/Lrespectively. However, to achieve maximal results, multipleconcentrations should be made that are, for example, 10% higher andlower in concentration of L-Cysteine and sodium nitrite, where eachconcentration is tested against the specific enzyme lot to be used todetermine the best concentration for use with the particular enzyme lot.A desired concentration provides an accurate, reliable, and detectablereadout. For example, a desired concentration provides minimal falsenegative and false positives. In preferred embodiments, where the testis to detected an alcohol concentration of 0.04%, there are not falsepositives when a 0.000 control is used, no more than 15% false positiveswhen 0.016 is used, and no more than 0.01% false negative results when0.064 is used. The water is added to a suitably sized vessel, followedby addition, with stirring, of the potassium EDTA, L-Cysteine, andcitric acid until completely dissolved. The pH of the mixture isadjusted to approximately 6.4 (e.g., using sodium hydroxide and HCl asnecessary). Once the pH is adjusted, sodium nitrite and potassium iodideare added and the mixture is stirred until everything is dissolved. Thesolution should be clear in color.

The third submix comprises water (RO/DI water) (20 g), sodium phosphate,dibasic, heptahydrate (134 mg), alcohol oxidase (80 KU), peroxidase (48KU), catalase (45 KU), and STABILGUARD (130 ml) (SurModics, Inc., EdenPrairie, Minn.). The water is added to a suitably sized vessel, followedby addition, with stirring, of the sodium phosphate dibasic,heptahydrate. The pH of the solution is adjusted to approximately 7.0.While gently stirring the phosphate buffer solution, the peroxidase isadded and slowly stirred until it is dissolved (approximately 10minutes). This solution is the peroxidase mixture. In a separate vessel,the alcohol oxidase is added and slowly stirred while the peroxidasemixture is added. Next, the catalase and STABILGUARD are added. Themixture is gently stirred for approximately 30 minutes. The solutionshould be a clear, cherry color.

In a vessel large enough to hold all three submixes, the submixed areadded together and stirred until completely mixed. Lactitol (50 g) isadded and thoroughly mixed until dissolved. The solution should beyellow and slightly cloudy. The solution is then mixed gently at roomtemperature for 90 minutes as an incubation period. The solution shouldbe used within 6 hours of preparation. To use, the solution is appliedto a desired test assay (e.g., applied to an absorptive material on atest strip). For example, the reaction mixture may be applied to a thinstrip of filter paper and dried (160° F.) to create a test strip.Application and drying are preferably conducted in a low humidityenvironment (e.g., ambient humidity of less than 5%, preferably lessthan 3%, and most preferably less than 2%) to facilitate fast drying andmaintain optimal reactivity and performance of the assay tests. In somepreferred embodiments, all manufacturing steps are carried out in a lowhumidity environment. In some embodiments, formulations are made asabove, but without catalase, nitrite, or citrate.

Each of the components present on the test strip are non-toxic andnon-carcinogenic. Each are found as food ingredients, approved foodadditives or substances normally encountered in the diet or producednormally in the body. Amounts present in the testing pad (e.g., 133micrograms of potassium iodide, 0.2 micrograms of sodium nitrite, 1.3micrograms of EDTA, sodium salt, 4.7 micrograms of cysteine, 123micrograms of citric acid, 533 micrograms lactitol, and 0.6 microgramsdisodium phosphate), only a portion of which would be solubilized andingested, represent small fractions of the amounts encountered in thedaily diet and from other sources such as in pharmaceutical products.

This reaction site provides a stable, accurate, inexpensive, non-toxic,non-irritant, non-carcinogenic on/off chemistry for use in alcoholdetection. The reaction time is less than two minutes and remainspositive for at least twenty additional minutes.

B) Glucose

The present invention provides non-invasive glucose tests. For example,the present invention provides oral glucose tests for monitoring glucoselevels from saliva. The glucose levels in saliva correlate with bloodglucose levels and can be used, whether quantitative or qualitative, inthe management of diabetes. Currently four billion dollars a year isspent on glucose testing for diabetics throughout the world. It isestimated that there are 16 million diabetics in the United States andmany more throughout the world. However, only 8 million of the diabeticsin the United States have been diagnosed with the disease. Approximately7 million of these diagnosed individuals are Type 2 diabetes and 1million are Type 1 diabetes. Type 1 diabetics must constantly measureand manage their glucose levels and must take insulin. Failure tocorrectly manage glucose levels in both Type 1 and Type 2 diabeticscould result in death. However, such negative consequences are morelikely and occur faster in Type 1 diabetics.

Current accurate products for measuring glucose levels involved taking ablood sample (e.g., pricking a finger) and having blood glucose measuredusing a portable electronic meter. Other expensive but accurate andnon-invasive products are being developed largely for Type 1 diabeticsincluding techniques such as infra-red scanning. Less accurate urinetests are available for Type 2 diabetics. Because of the nature ofcurrent diagnostics, Type 2 diabetics do not test themselves very often.Hence, there is a market need for Type 2 diabetics to test themselveswith an inexpensive, qualitative, non-invasive product. The assay testsof the present invention meet these needs.

In some embodiments of the present invention, the glucose test assay isa qualitative assay, indicating the presence of glucose at or above aparticular threshold level. Qualitative assays find use by diabetics inmonitoring glucose levels, wherein the information is used in makingdietary and/or medical decisions. In other embodiments, the glucose testassay is a quantitative assay, wherein a particular concentration ofglucose is determined.

Glucose assay tests of the present invention may be used with thedelivery systems of the present invention, provide durability, improvedshelf-life, and ease-of-use.

The non-toxic, non-irritant, non-carcinogenic reactions means discussedabove for alcohol detection finds use in the glucose assay tests.Glucose oxidase is supplied in place of alcohol oxidase. Glucose oxidasereacts with glucose present in the saliva sample to generate gluconicacid and hydrogen peroxide. The hydrogen peroxide, like that in thealcohol detection assay, stimulates a color response. Thus, in somepreferred embodiments, the present invention provides a glucose assaytest comprising glucose oxidase and a non-toxic chromogen (e.g.,potassium iodide). In other embodiments, the reaction means compriseshexokinase which reacts with glucose to form glucose-6-phosphate withthe concomitant conversion of NADP⁺ to NADPH. The NADP⁺/NADPH conversionmay be coupled to a color detection system (See e.g., U.S. Pat. Nos.5,032,506 and 5,036,000, herein incorporated by reference in theirentireties). In preferred embodiments, the reaction means containing theoxidase and chromogen is present in a reactive pad on the end of a teststrip, wherein the pad is placed directly in the mouth. Additionalsystems, chemistries, and detection modes for carrying out glucosedetection are described in U.S. Pat. Nos. 6,102,872, 3,964,871,5,217,691, 5,140,985, 6,194,224, 5,179,288, 5,714,341, 5,989,917,4,476,222, and 5,912,139, herein incorporated by reference in theirentireties.

C) Other Assay Tests

1) Prostate-Specific Antigen (PSA)

The present invention provides non-invasive, inexpensive PSA tests. Forexample, the present invention provides oral PSA tests for monitoringPSA levels from saliva. High PSA levels in saliva correlate withprostate cancer, a noncancerous (benign) enlargement of the prostate(benign prostatic hypertrophy), and infected or injured (trauma)prostate. Every male over the age of 50 in the world should be testedregularly for prostate cancer. Current methods for testing for prostatecancer are two-fold: either an invasive rectal exam or expensive bloodwork. Because tests are invasive and expensive, many people are nottested regularly, or at all.

In some embodiments of the present invention the reaction means of theassay tests of the present invention contains an anti-PSA antibody(i.e., any immunoglobulin molecule that specifically interacts with PSAor a unique epitope of PSA). The reaction site is placed into the mouthto allow binding free PSA in the saliva to the anti-PSA antibody. Thebound complex is then detected using any system of method known in theart. In some embodiments, detection requires the use of a detectionapparatus. Systems and methods for PSA detection are described in U.S.Pat. Nos. 5,614,372 and 6,200,765, herein incorporated by reference intheir entireties.

2) Ketones

The present invention provides non-invasive, inexpensive ketone tests(i.e., detection of ketone bodies such as acetone, acetoacetic acid andβ-hydroxybutyric acid). For example, the present invention provides oralketone tests for monitoring ketone levels in saliva. Approximately 2.5million people world-wide are performing diets where ketone levelsshould be monitored. Also, there are approximately 1-5 million highperformance athletes that should regularly measure ketone levels. Ifdieters' or athletes' ketone levels increase too high, they can go intoacidosis and their bodies process muscle tissue instead of fat. Thequalitative and/or quantitative measurement of ketone concentrations isimportant because of the relationship between elevated serum ketonelevels and clinical conditions such as diabetes, disorders of thedigestive organs, renal insufficiency, uremia and malignant carcinoma.In the course of these disorders, ketone bodies pass into the bloodstream and a state of metabolic acidosis (ketosis) occurs. Monitoringfor the onset of ketosis is of particular importance in the maintenanceof diabetics because the occurrence of ketosis may indicate the need formodification of insulin dosage or other disease management.

The non-toxic, non-irritant, non-carcinogenic reactions means discussedabove for alcohol and glucose detection finds use in the ketone assaytests. In some embodiments, the ketone is reacted with enzymes to formoxidized or reduced products (e.g., hydrogen peroxide, NADP⁺ or NADPH)that can be detected in the systems described above. For example, insome embodiments, the reaction means comprises a dehydrogenase (e.g.,3-hydrocybutric dehydrogenase) which causes NADP⁺ NADPH conversion (Seee.g., U.S. Pat. No. 5,618,686, herein incorporated by reference in itsentirety). The NADP⁺/NADPH conversion may be coupled to a colordetection system (See e.g., U.S. Pat. Nos. 5,032,506 and 5,036,000,herein incorporated by reference in their entireties). Additionalsystems, chemistries, and detection modes for carrying out ketone bodydetection are described in U.S. Pat. Nos. 3,880,590, 4,440,724,4,405,721, 4,184,850, 4,097,240, 3,212,855, 4,970,172, and 4,147,514,herein incorporated by reference in their entireties.

Because glucose and ketone levels are both relevant to diabetics, insome embodiments, the present invention provides assay tests thatsimultaneously detect glucose and ketone levels.

3) Cortisol

The present invention provides non-invasive, inexpensive cortisol tests.For example, the present invention provides oral cortisol tests formonitoring cortisol levels in saliva. Cortisol is a hormone that hasspecific correlation to mood management and behavior. Low cortisollevels likely result in a person being in a bad mood and also may havean impact on blood pressure. Currently 1.5 million cortisol tests areperformed each year.

In some embodiments of the present invention the reaction means of theassay tests of the present invention contains an anti-cortisol antibody(i.e., any immunoglobulin molecule that specifically interacts withcortisol or a unique epitope of cortisol). The reaction site is placedinto the mouth to allow binding free cortisol in the saliva to theanti-cortisol antibody. The bound complex is then detected using anysystem of method known in the art. In some embodiments, detectionrequires the use of a detection apparatus. Systems and methods forcortisol detection are described in U.S. Pat. Nos. 5,910,575, and4,311,690, herein incorporated by reference in their entireties.

Example 1 Toxicity/Irritation Test

Ten healthy male Golden Syrian Hamsters are assigned to two groups offive animals/group. Group 1 is dosed with aliquots of solutioncontaining the detection assay component at a concentration equivalentthat intended for use in the detection assay, as well as, one or morediluted or concentrated samples (e.g., 2, 5, 10, and 100-fold dilute andconcentrated compared to the intended use concentration) and Group 2 isdosed with saline. Prior to study initiation, the left cheek pouch ofall animals is abraded using emery paper (everted and three areas of themucosa abraded with three firm strokes of fine grit emery paper). Onlyanimals with scalar notations of “zero” for erythema and edema for bothleft and right pouches are used. Following site preparation, 0.1 ml ofthe test or placebo is gently applied to the buccal mucosa of the leftand right cheek pouch using a 1 cc tuberculin syringe. This procedure isrepeated twice daily for four consecutive days and once on the fifthconsecutive day. Prior to the first and each subsequent treatment, foodparticles are cleared from the left and right cheek pouches withapproximately 20 ml of distilled water (room temperature) using asyringe equipped with a 12 gauge, 2 inch long blunt needle covered withrubber tubing. The left and right pouch are everted and examined forcleanliness. If food particles remain, the rise is repeated. The cleaneverted pouch is examined and scored with the aid of an incandescenthigh intensity light.

Erythema and edema are scored prior to each treatment and prior tonecropsy in the left and right pouch of each animal. The animals areobserved once daily for mortality, toxicity and pharmacologic effects.Body weights are recorded pretest and at termination. Following the lastobservation of day 5, the animals are humanely sacrificed and the leftand right pouches are preserved in 10% neutral buffered formalin forhistopathologic evaluation. The Group Average Mucosal Irritation Scoresare calculated for both intact and abraded pouches.

The following scoring scale is used:

ERYTHEMA & ESCHAR FORMATION VALUE Natural pink condition of mucosa 0Well-defined erythema 1 Moderate to severe erythema 2 Severe erythema(beet redness) 3 Loss of color (blanching of mucosal, etc.) 4

EDEMA FORMATION VALUE Normal condition (note folds in mucosa) 0 Slightedema (edges of area well-defined by definite raising) 1 Moderate edema(area raised approximately 1 mm) 2 Severe edema (raised >1 mm &extending beyond exposure 3 area) Blistering 4

For each group at each time period, the individual erythema scores areadded and the sum divided by the number of scores added to obtain theGroup Average Erythema Score for each time period for the intact andabraded sites. This procedure is repeated using the edema scores toobtain the Group Average Edema Scope for each site at each time period.

For each day, the group average erythema scores for each time period andthe group average edema scores for each time period are added and thissum divided by four (4) to obtain the Daily Group Average MucosalIrritation Score.

In some embodiments, only materials that have an average score of lessthan two for each of the erythema and edema scores are used in detectionassays. Preferably, the average score is less than 1 (i.e., non-toxic,non-irritant). In particularly preferred embodiments, the average scoreis less than 0.8 or even more preferably, less than 0.5. Any othersuitable testing procedure may also be used (See e.g., ToxicologyTesting Handbook: Principles, Applications, and Data Interpretation, ed.Jacobson-Kram and Keller, 2001).

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled in therelevant fields are intended to be within the scope of the followingclaims.

1. (canceled)
 2. A method for detecting the presence of ethanol in abiological sample from a subject, comprising: a) contacting an assaytest comprising a reaction site comprising i) a potassium iodidechromogen, ii) an alcohol oxidase enzyme, and iii) a peroxidase enzymewith a biological sample from a subject under conditions such that saidsample contacts said reaction site; and b) detecting the presence orabsence of a visibly detectable signal in said reaction site, whereinsaid visibly detectable signal comprises a color change that occurs at abiological sample ethanol concentration at or above a threshold leveland wherein no color change occurs below said threshold level.
 3. Themethod of claim 2, wherein said threshold level is equivalent to a bloodalcohol concentration of 0.04%.
 4. The method of claim 2, said assaytest comprises a test strip.
 5. The method of claim 4, wherein said teststrip comprises an absorbent material, wherein said reaction site islocated within said absorbent material.
 6. The method of claim 2,wherein said color change is detectable by the human eye.
 7. The methodof claim 2, wherein said biological sample is a fluid.